It is known that cancerous or otherwise abnormal cells of identical histopahtological type show a wide range of responsiveness to particular drug therapies among individual patients. Predictive techniques, similar to the culture and sensitivity assays used for the management of microbial infections, would be of great assistance in selecting effective chemotherapy for individual cases.
Without individualized anti-cancer drug regimens, practitioners are forced to rely on past experience or reports on similar cell disorders or trial-and-error procedures. With the increasing number of anti-cancer agents available and the limited time often available for modifying doses or agents, the task of selecting the optimal regimen, without the aid of predictive assays, is very difficult.
A number of predictive systems have been proposed. See, for example, Salmon et al., "Quantitation of Differential Sensitivity of Human Stem Cells to Anti-Cancer Drugs," Vol. 298 New England Journal of Medicine pp. 1321-1327 (1978). Typically, the prior art techniques involve the cloning of single cell suspensions from biopsy specimens in soft agar after brief exposure to particular anit-cancer drugs. See also, Buick et al., "Development of an Agar-Methyl Cellulose Clonogenic Assay for Cells in Transitional Cell Carcinoma of the Human Bladder," Vol. 39 Cancer Research pp. 5051-5056 (1979) and Von Hoff et al., "Direct Cloning of Human Malignant Melanoma in Soft Agar Culture," Vol. 50 Cancer pp. 696-701 (1982), for further details on agar culture techniques.
Various difficulties limit the usefulness of agar culture studies for predicting the effectiveness of cytoxic agents against abnormal cells. Only a small fraction of biopsied cancer cells grow in soft agar. For example, when cell suspensions from myeloma specimens are plated in agar, plating efficiencies of 1:1000 are not uncommon. Thus, for statistically significant results comparing different drugs at different doses, large number of cells are required. It is also not certain that colonies formed in agar will be derived from the most malignant tumor cells. Moreover, agar techniques typically limit drug exposure to a relatively brief period (i.e., one hour) prior to plating while the cell is suspended in a physiological solution. Neither the exposure technique nor the subsequent growth in agar accurately mimic in vivo conditions. Additionally, the time required for evaluation is long (i.e., 14 to 30 days) compared to the often urgent need to establish a protocol for therapy. Finally, measurements of drug sensitivity by counting cell colonies can be subjective, statistically inaccurate and time consuming.
Another predictive system which has been proposed for chemotherapy studies involves the use of cell cultures grown in an artificial organ made of a matrix of synthetic capillaries. Quartles et al., "Hemodialysis-Matrix Perfusion Culture System: A New Technique for Studying Chemotherapeutic Activity Tumor Cells," Vol. 16 In Vitro 246 (1980), report the effect of one anti-cancer agent on tumor cells grown in an artificial organ system (Amicon-Vitafiber (R)). Following exposure to the drug, the cultured cells were removed from the organ and assayed for total and viable cells, colony forming ability and growth in soft agar.
Synthetic capillary systems have advantages over soft agar techniques in presenting a culture more similar to the in vivo environment and permitting the introduction of drugs into the culture via the capillaries in a fashion more like the perfusion of active agents in a patient. For a review of capillary cultures, generally, see Schratter, "Cell Culture with Synthetic Capillaries," Vol. XIV Methods in Cell Biology pp. 95-103 (1976), herein incorporated by reference.
The capillary technique for studying chemotherapeutic activity reported by Quartles, supra, is still subject to many of the same problems that limit the usefulness of agar studies. Quartles and his co-workers had to remove the cells from the capillary system in order to count total and viable cells. In practice, removing cells without damage from a capillary matrix is an ardent task. Typically, the cells are removed from the capillary matrix by enzyme treatment but this treatment can be more effective on dead cells than on living cells and quantitiative results are difficult to obtain. Moreover, the culture is lost after enzyme treatment and can not be used again.
Additionally, once the cells are removed, counting viable cells under a microscope again can be subjective and inaccurate and certainly is time consuming. A drug sensitivity test which relies on visual observations of live cells is very unlikely to find widespread clinical application.
There exists a need for simple, efficient methods and apparatus for predicting the in vivo responsiveness of cancerous and otherwise abnormal cells to the therapeutic agents. The predictive culture system should be easily innoculated while cell growth and drug exposure should mimic closely the human environment. More importantly, the drug sensitivity should be quantifiable by a simple and accurate method in a relatively short time and preferably in a way that would permit a clinician to obtain a reading on the effectiveness of a particular agent without destruction of the culture so that the effects of a multi-step protocol (i.e., varying in agents or doses) can be measured sequentially.